Unidad 1, sección 1.1, subsecciones 1.1.1 y 1.1.2

VO2peak was the only outcome from this study obtained from the metabolic cart; this was used to help determine when the participant had reached maximum exertion.

Incremental Cycle Ergometer Form. The research team completed the Incremental Cycle Ergometer form during each maximal exercise test to track heart rate and level of

perceived exertion at each stage of testing. Heart rate and total time of maximal exercise test at protocol termination were the study outcomes recorded on this form.

Polar Heart Rate Monitors. Each subject wore a Polar H1 Heart Rate Sensor (Kempele, Finland) while completing both of the maximal exercise testing sessions. These monitors were strapped to patients around their chest, and could be fitted comfortably under the shirt of the participant. The monitor transmitted data via Bluetooth to a synced watch, including heart rate of the participant throughout the study, which was used as an outcome in this study.

Borg Rate of Perceived Exertion Scale. The Borg Rate of Perceived Exertion (RPE) Scale measured the participants’ perception of the difficulty of the maximal exercise test. Participants were asked to rate their level of exertion on a scale from 6 (no exertion) to 20 (maximal exertion) 30 seconds before the termination of each stage of the maximal exercise test. The Borg RPE Scale was used along with the VO2peak data, maximal heart rate, and lactate levels to corroborate when the participant had reached their maximum exertion level during the exercise test.

Blood Processing and Analysis Instruments

Centrifuge. After blood collection, a Sorvall ST 8R Centrifuge (Waltham, MA) was used to obtain serum isolates for analysis. The high centripetal force generated by the centrifuge pulls the elements of the blood with more mass, such as erythrocytes, to the bottom of the test tube.

As a result, the separated serum isolate is left at the top of the tube for use. There were no study outcomes obtained from the centrifuge, as its purpose was to prepare the sample for storage.

Hemoglobin/Hematocrit Analyzer. Prior to spinning the blood samples, Hemoglobin and hematocrit were measured using a HemoPoint H2 hemoglobin analyzer (Boerne, TX) using whole blood. This analyzer uses the photometric azide methemoglobin method to reliably measure the amount hemoglobin protein in the blood plasma. Hemoglobin amount and hematocrit were derived from this analysis to control for plasma volume shift.

UCH-L1 Analysis. All blood samples were shipped to Banyan Biomarkers, Inc. (San Diego, CA). The blood was shipped priority overnight, and was completely covered in dry ice for the shipping process. Banyan thawed the serum samples, and then performed a sandwich ELISA. Blood specimens were applied to a 96-well plate that had been pre-coated with an antibody specific to UCH-L1. The plate was then washed to remove any unbound material. Banyan then added a second antibody that was specific to UCH-L1 to aid with detection (creating the “sandwich”) before repeating the wash step. A substrate that reacts with an enzyme attached to the detection antibody was then added to the plate, producing a chemiluminescent signal that could be measured electronically and is proportional to the

amount of biomarker present. Biomarker concentration (pg/ml) was determined from comparing the chemiluminescent signal strength with a standard curve created from a series of samples with known concentrations of UCH-L1 that were run on the same 96-well plate. All samples were analyzed in triplicate (intra-plate CV = 5%, inter-plate CV = 5%), and the values used in the results were the average of the three samples.

Procedure

Participants were introduced to the equipment and procedures during an orientation session to limit any potential learning or familiarization effects. Participants then returned for two, identical clinical testing sessions. Healthy participants enrolled into the study at an arbitrary start date and completed with two clinical testing sessions within 10-14 days of each other. Concussed participants were enrolled into the study within 3-10 days of their injury. Concussed participants completed the first clinical testing sessions as close to three days following injury as possible and the second clinical testing sessions following 24 consecutive hours without

concussive symptoms.

Orientation Session

Prior to testing sessions, participants completed an orientation session to ensure sufficient cardiovascular health to successfully complete the maximal exercise testing, as well as to orient them to the equipment and the timing and intensity of the maximal exercise testing protocol. All participants completed the Physical Activity Readiness Questionnaire (PAR-Q), self-reported a general medical history, and underwent a 10-lead EKG, all of which were read by an independent medical monitor. The independent medical monitor provided medical

clearance for each participant to verify that the participants’ cardiovascular health was sufficient to perform maximum exercise testing without any serious health risks. Any participants not receiving medical clearance (n=4) were excluded from study participation. Participants were then introduced to and fitted for the facemask equipment associated with the metabolic cart and cycle ergometer. Ensuring proper fit to the equipment was necessary to ensure minimal

protocols. Healthy participants were given an abbreviated version of the maximal exercise test to familiarize them with the exercise protocol. Healthy participants were allowed to pedal for five minutes at a comfortable pace with no resistance, and then completed the first two stages of the maximal exercise test. Concussed participants were not given the abbreviated version maximal exercise test due to safety reasons, but were fitted for all equipment. Lastly, the timing, volume, and number of blood draws completed during each testing session was explained to the

participants, who were then given a chance to ask any questions.

Clinical Testing Session 1 and 2

Following the orientation session and medical clearance from the independent medical monitor, participants were scheduled for the first of two, identical clinical testing sessions. Upon arriving for a clinical testing session, participants completed a blood draw in which one 10ml EDTA and one 10ml SST tube were drawn using standard phlebotomy procedures. Following the first blood draw, participants were outfitted with a heart rate monitor (Polar, Kempele, Finland), then asked to sit quietly and rest for five minutes. Participant’s vital signs (heart rate and blood pressure) were taken following this rest period.

Table 3: Maximal Exercise Test Protocol for Concussed Participants

0:00 – Begin Pedaling at 25 W workload 2:00 – Workload increased to 50 W 4:00 – Workload increased to 75 W 6:00 – Workload increased to 100 W 8:00 – Workload increased to 125 W 10:00 – Workload increased to 175 W

Termination – Volitional fatigue or exacerbation of concussion symptoms

After obtaining vital signs, the participant completed the maximal exercise test. The maximal exercise testing protocol was identical for all